NTRK fusions lead to the expression of oncogenic chimeric TRK proteins with constitutively activated kinases, similar to ALK and ROS1 fusions. Most importantly, NTRK fusions are highly actionable in the clinic. First- generation TRK kinase inhibition (larotrectinib) results in rapid and durable histology-agnostic responses, with an objective response rate of 76% across a wide variety of solid tumors (Drilon et al., NEJM, provisionally accepted). Despite these impressive results, little is known about the biology of NTRK fusions, and resistance to 1st-generation therapy ultimately develops. While 2nd-generation inhibitors address on-target resistance, the development of acquired resistance to these drugs likewise represents a challenge. The main objectives of this proposal are to elucidate signaling pathways that mediate the activation and transformative capacity of TRK fusion proteins, and to identify mechanisms of intrinsic or acquired resistance to TRK inhibitors. For this project, we plan to leverage (1) our leadership in ongoing TRK inhibitor clinical trials, (2) our prior experience in identifying and characterizing resistance mechanisms to targeted therapy, and (3) our creation of a multidisciplinary ?TRK team? of scientists, pathologists, and clinicians to study TRK biology. In order to shed light on signaling pathways and/or gene expression patterns that mediate TRK fusion kinase activity, we will perform an unbiased global proteomic/transcriptomic screening using patient-derived models treated with 1st- or 2nd-generation TRK inhibitors. Candidate proteins/pathways involved in TRK-mediated tumorigenesis will be validated both genetically and pharmacologically. To determine mechanisms of resistance to 1st-generation (larotrectinib and entrectinib) and 2nd-generation (LOXO-195 and TPX-0005) inhibitors in the clinic, we will perform comprehensive characterization of paired pre- and post-treatment biopsies from patients with NTRK-rearranged solid tumors treated at our institution. Tumors will be characterized by targeted capture-based exome sequencing (MSK-IMPACT), anchored multiplex PCR (MSK Archer Solid Tumor Panel), and pan-TRK IHC; in addition, serial plasma profiling (ddPCR and hybrid capture) will be performed in patients on TRK inhibitor therapy. Building on our prior identification of convergent, on-target resistance (solvent-front mutations), we identified off-target resistance mediated by MAPK pathway reactivation (NRAS/BRAF/GNAS mutations) that may be amenable to combination therapy. For 2nd-generation inhibitor resistance, we have already identified a novel compound NTRK mutation. These efforts will be complemented by the routine creation of NTRK-rearranged patient-derived cell lines and xenografts, and engineered models (transduced primary cell lines/CRISPR-modified). In addition to exploring downstream signaling as previously described, therapeutic strategies will be explored in vitro and in vivo for both on-target resistance (2nd-generation TKI switching), and off-target resistance (combination therapy, e.g. TRK and MEK inhibition). When feasible, candidate strategies will then be explored in the clinic.